Peripheral blood mononuclear lymphocytes (PBLs) can be stimulated to develop lytic activity against fresh tumor cells, as well as several natural killer (NK) resistant targets, such as Daudi and HL60, after relatively short-term (3-5 days) culturing in the presence of recombinant interleukin-2 (IL-2). See, for example, M. Lotze et al., Cancer Res., 41, 4420 (1981); C. Grimm et al., J. Exp. Med., 155, 1823 (1982); and E. A. Grimm et al., "The Lymphokine-Activated Killer Cell Phenomenon: In Vitro and In Vivo Studies," in Interleukins, Lymphokines and Cytokines, S. Cohen and J. Oppenheim, eds., Academic Press, New York, p. 739 (1983). This function has been termed lymphokine activated killer (LAK) activity.
Initial reports suggested that precursors of cells with LAK activity did not express the T cell receptor as determined by anti-CD3 binding. See, E. A. Grimm et al., J. Exp. Med., 157, 884 (1983). More recent reports have demonstrated that effector cells obtained from short-term culturing with IL-2 (2-5 days) are a CD3.sup.- population of cells that express the NK markers CD16 and/or CD56. The CD3.sup.+ cells from such cultures have low lytic activity against NK-resistant targets. Thus, the CD3.sup.- population, with the NK markers CD16 and/or CD56, is responsible for the great majority of the LAK activity in PBL cultures. That is, CD3.sup.- cells appear to be the classical NK effector cells. See, for example, J. R. Ortaldo et al., J. Exp. Med., 164, 1193 (1986); S. Ferrini et al., J. Immunol., 138, 1297 (1987); K. Itoh et al., J. Immunol., 136, 3910 (1986); and J. H. Phillips et al., J. Exp. Med., 164, 814 (1986).
Reversible induction of NK activity in cloned cytotoxic lymphocytes in response to IL-2 and interferon (IFN) has been reported. See, C. G. Brooks, Nature, 305, 155 (1983). Furthermore, the generation of large numbers of cells with LAK activity using relatively long-term cultures (10-30 days) of PBLs stimulated with the anti-CD3 monoclonal antibody (MoAb) OKT3, in combination with IL-2 has been reported (CD3-LAK cells or T-AK cells). See, A. C. Ochoa et al., J. Immunol., 138, 2728 (1987). The effector cells in these long-term IL-2 and OKT3 cultures include CD3.sup.- cells, as well as a CD3.sup.+ population that is both CD4 and CD8 negative, and expresses the .gamma..delta. chains of the T cell receptor. In contrast, the effector cells in short-term IL-2 and OKT3 cultures (2-5 days) are predominantly CD3.sup.- cells.
Numerous studies have shown that very little LAK activity appears to be mediated by the classically described CD4.sup.+ or CD8.sup.+ T cells. Furthermore, CD4.sup.+ or CD8.sup.+ cells isolated from cultures of mixed PBL populations, which are activated with an antibody to a lymphocyte surface receptor, such as the anti-CD3 monoclonal antibody OKT3, and continuously cultured with IL-2, do not develop significant levels of NK or LAK activity, as determined immediately upon their isolation from the total population. See, for example, A. C. Ochoa et al., Cancer Res., 49, 963 (1989). For example, at an effector to target ratio of about 30:1, i.e., a ratio of the number of T cells capable of mediating cytotoxicity to the number of tumor cell line targets, the cytotoxicity of CD4.sup.+ or CD8.sup.+ subsets is no more than about 15-20%.
It has been noted that when PBLs are stimulated in mixed lymphocyte culture (MLC), the CD4.sup.+ cells are minimally cytotoxic. Furthermore, when the CD4.sup.+ population is stimulated in MLC in the absence of other T cells, they develop greater cytolytic activity. See, E. L. Reinherz et al., Proc. Natl. Acad. Sci. USA, 76, 4061 (1979). However, this cytotoxicity is antigen specific, and does not involve tumor killing activity.
It has also been recently shown that CD8.sup.+ CD11b.sup.+ cells can develop LAK activity. In this specific situation, the CD8.sup.+ T cells were isolated from the PBL population before the initiation of culture in the presence of IL-2 alone. However, this method did not involve anti-CD3 MoAb stimulation; however, this method did involve separating the T cells with sheep red blood cells, which in itself can produce a stimulating signal through the CD2 receptor. Thus, NK cells, which express the CD2 receptor, can also be activated. See, U. Dianzani et al., Eur. J. Immunol., 19, 1037 (1989).
CD4.sup.+ and CD8.sup.+ cells cultured in the presence of IL-2 alone have been shown to express the lytic machinery, but LAK activity was not demonstrated nor was cell growth reported. See, M. J. Smyth et al., J. Exp. Med., 171, 1269 (1990). Finally, it has been shown that tumor infiltrating lymphocytes (TILs), which appear to be effective in the treatment of solid tumors, are primarily CD8.sup.+. See, for example, S. Shu et al., J. Immunol., 139, 295 (1987); and A. Belldegrun et al., Cancer Res., 48, 206 (1988).
The identification of cells that can mediate cytotoxicity, e.g., LAK activity, is important both for an understanding of the interactions of the immune system as well as for the potential development of effective methods of immunotherapy. One of the limitations of current LAK therapies for the treatment of tumors is that LAK cells appear to be transported via the reticuloendothelial system which, in some cases, limits the accessibility of LAK cells to certain tumors. See, for example, A. A. Maghazachi et al., J. Immunol., 141, 4039 (1988). T cells, on the other hand, circulate through the lymphatic system and provide greater accessibility to most tumors.
While most NK and LAK activity in cultures stimulated with IL-2 alone or IL-2+anti-CD3 appears not to be mediated by CD4.sup.+ or CD8.sup.+ cells, what has been needed is to determine if these T cells, under the appropriate conditions, could develop high cytotoxicity, e.g., specific or nonspecific lytic activity. Thus, what is needed is a method for the stimulation of high cytotoxicity, preferably high nonspecific lyric, e.g. NK or LAK, activity in T cells, which can provide antitumor therapeutic efficacy.